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Identification and Capsular Serotype Sequetyping of Streptococcus Pneumoniae Strains

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Identification and Capsular Serotype Sequetyping of Streptococcus Pneumoniae Strains bioRxiv preprint doi: https://doi.org/10.1101/415422; this version posted September 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Identification and capsular serotype sequetyping of Streptococcus pneumoniae strains Lucia Gonzales-Silesa,b*, Francisco Salvà-Serraa,b,c,d, Anna Degermana, Rickard Nordéna, Magnus Lindha, Susann Skovbjerga,b, Edward R. B. Moorea,b,d. a Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden b Centre for Antibiotic Resistance Research (CARe), University of Gothenburg, Gothenburg, Sweden c Microbiology, Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain d Culture Collection University of Gothenburg (CCUG), Department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden * Corresponding author E-mail address: [email protected] (LG) Post address: Guldhedsgatan 10A 41346 Gothenburg, Sweden bioRxiv preprint doi: https://doi.org/10.1101/415422; this version posted September 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT Correct identification of Streptococcus pneumoniae (pneumococcus) and differentiation from the closely related species of the Mitis group of the genus Streptococcus, as well as serotype identification, is important for monitoring disease epidemiology and assessing the impacts of pneumococcal vaccines. In this study, we assessed the taxonomic identifications of 422 publicly available genome sequences of S. pneumoniae, S. pseudopneumoniae and S. mitis, using different methods. Identification of S. pneumoniae, by comparative analysis of the groEL partial sequence, was possible and accurate, whereas S. pseudopneumoniae and S. mitis could be misclassified as S. pneumoniae, suggesting that groEL is unreliable as a biomarker for differentiating S. pneumoniae from its closest related species. The genome sequences of S. pneumoniae and S. pseudopneumoniae fulfilled the suggested thresholds of average nucleotide identity (ANI), i.e., >95% genome sequence similarity to the sequence of respective type strains for identification of species, whereas none of the S. mitis genome sequences fulfilled this criterion. However, ANI analyses of all sequences versus all sequences allowed discrimination of the different species by clustering, with respect to species type strains. The in silico DNA-DNA distance method was also inconclusive for identification of S. mitis genome sequences, whereas presence of the “Xisco” gene proved to be a reliable biomarker for S. pneumoniae identification. Furthermore, we present an improved sequetyping protocol including two newly-designed internal sequencing primers with two PCRs, as well as an improved workflow for differentiation of serogroup 6 types. The proposed sequetyping protocol generates a more specific product by generating the whole gene PCR-product for sequencing, which increases the resolution for identification of serotypes. Validations of both protocols were performed with publicly available S. pneumoniae genome sequences, reference strains at the Culture Collection University of Gothenburg (CCUG), as well as with clinical isolates. The results were compared with serotype identifications, using real-time Q-PCR analysis, as well as the Quellung reaction or antiserum panel gel-precipitation. Our protocols provide a reliable diagnostic tool for taxonomic identification as well as serotype identification of S. pneumoniae. Keywords: Streptococcus pneumoniae; serotype; sequetyping; Mitis group bioRxiv preprint doi: https://doi.org/10.1101/415422; this version posted September 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. INTRODUCTION Streptococcus pneumoniae (pneumococcus) causes invasive and non-invasive disease, including pneumonia, meningitis, sepsis, otitis media, sinusitis, among others, particularly in children under the age of 5 years and the aged (Johnson et al., 2010), leading to approximately a million deaths annually in children aged less than 5 years, globally (Collaborators, 2017). A characteristic feature and the main virulence factor of S. pneumoniae is the polysaccharide capsule that enables the bacterium to evade host defence mechanisms (Nelson et al., 2007) and which is the basis for epidemiological categorization of pneumococcal isolates and strains into serotypes and serogroups (Geno et al., 2015). To date, 97 different capsular serotypes within 46 serogroups of S. pneumoniae have been identified on the basis of the biochemical structure of the capsular polysaccharide (Geno et al., 2015). Several pneumococcal vaccines, which differ according to the polysaccharide capsule composition, have been developed. The first pneumococcal conjugate vaccine (PCV), licensed in 2000, covered 7 serotypes (PCV7: 14, 6B, 19F, 23F, 4, 9V, 18C) (Hicks et al., 2007), followed by PCV10 (PCV7 serotypes plus serotypes 1, 5, and 7F) in 2009 (Esposito and Principi, 2015), PCV13 (PCV10 serotypes plus serotypes 3, 6A, and 19A) in 2010 (Geno et al., 2015). A 15-valent conjugate vaccine is currently in clinical trials, and includes also serotypes 22F and 33F (LeBlanc et al., 2017). The pneumococcal polysaccharide vaccine (PPSV23) protects against 23 different capsular types (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), and covers a high percentage of the types found in pneumococcal bloodstream infections. The vaccine is widely used for adults who are considered to be at high risk, as well as in children older than 2 years and at increased risk for pneumococcal disease (Diao et al., 2016). The use of bioRxiv preprint doi: https://doi.org/10.1101/415422; this version posted September 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. the conjugate vaccines have significantly reduced the burden of pneumococcal disease in many populations. However, since vaccine introduction, “serotype replacement” has been observed, with increases in the proportions of invasive and non-invasive disease caused by pneumococcal serotypes not covered by the vaccines (Hicks et al., 2007; Weinberger et al., 2011). S. pneumoniae serotype 6C is an example of an opportunistic increase in an infectious pneumococcus through serotype replacement. Serotype 6C was described as a newly-recognized serotype in 2007 (Mavroidi et al., 2004) and appears to have been rare in pre-vaccination populations. However, since the introduction of PCV7, the incidence of serotype 6C in disease and carriage has increased in diverse populations, worldwide (Loman et al., 2013). PCV7 contains polysaccharide from the 6B serotype capsule and PCV13 later included capsular polysaccharide of serotype 6A, although current vaccines do not extend protection to serotype 6C, which likely has promoted the observed serotype replacement (Park et al., 2008). Such serotype transitions demonstrate the importance of maintaining surveillance programs and clinical protocols that are able to respond to the evolutionary plasticity of infectious disease. The classical serotyping method, the Quellung reaction, is based on the reaction of serotype- specific antisera with the corresponding capsule (Neufeld F, 1910). This method is time-consuming and costly, requiring live, cultivable bacteria, and a high degree of expertise, to the point that few laboratories are able to carry out the analyses. During the last decade, the nucleotide sequences of the capsule polysaccharide synthesis (CPS) loci (cps), harbouring the genes responsible for synthesis of the pneumococcal cell polysaccharide capsule, have been determined for all known serotypes. Accordingly, DNA amplification-based methods targeting specific capsular synthesis genes that allow differentiation of the serotypes have been developed, i.e., sequential multiplex bioRxiv preprint doi: https://doi.org/10.1101/415422; this version posted September 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. PCR and sequential real-time Q-PCR (Pai et al., 2006; Varghese et al., 2017). Recently, a PCR- amplification and DNA-sequence-based typing method, ‘sequetyping’, was described targeting the regulatory gene, cpsB, with a single multiplex PCR, enabling the amplifications of 84 serotypes and sequencing of PCR-products, differentiating 46 of the 93 serotypes recognized at that time (Leung et al., 2012). As an important practical step, before initiating pneumococcal serotype identification, it is critical to confirm the identification of S. pneumoniae and differentiate it from the other species of the Mitis group of the genus Streptococcus (Kawamura et al., 1995). The most closely-related species of S. pneumoniae are S. pseudopneumoniae and S. mitis. Sequencing of the 16S rRNA genes identifies a cytosine nucleotide at position 203 as a pneumococcal sequence signature, with an adenosine residue in all other species of the Mitis group (Scholz et al., 2012). Partial sequence determinations of individual metabolic ‘housekeeping’ genes, as a multi-locus
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